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Regular article |
a Inland Station, National Research Institute of Aquaculture, Tamaki, Mie 519-0423, Japan
b National Research Institute of Aquaculture, Nansei, Mie 519-0196, Japan
c Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 022-0101, Japan
ABSTRACT
Two distinct gonadotropins (GTHs) have been demonstrated in a number of teleost fishes. Although the physiological roles of GTHs have been extensively studied in salmonids, little is known about their biological functions in nonsalmonid fishes. In this study, to elucidate the role of GTH-I and GTH-II in reproduction, we cloned the
-glycoprotein subunit (GSU) and gonadotropin ß subunits (Iß and IIß) of red seabream using the 5'- and 3'-RACE methods and used these cDNA probes to reveal changes in mRNA levels of each subunit during sexual maturation of both male and female red seabream. The nucleotide sequences of GSU, Iß, and IIß are 629, 531, and 557 base pairs long, encoding peptides of 117, 120, and 146 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high homology with those of other teleosts. Northern blot analysis showed that Iß mRNA levels of males increase in association with gonadal development, whereas those of females remain low throughout sexual maturation, indicating sexual dimorphism in the expression pattern of Iß. In contrast, IIß mRNA levels of both sexes are maintained at high levels from the beginning of gametogenesis to spawning season. These results are different than those of salmonids and suggest that GTH-I may have important roles in male, but not female, gametogenesis. GTH-II may be involved in regulation of early and late gametogenesis in both male and female red seabream.
anterior pituitary, FSH, LH, seasonal reproduction
INTRODUCTION
Gonadotropins (GTHs), LH and FSH, are critical modulators of gametogenesis and gonadal steroidogenesis in almost all vertebrates, including teleosts. These glycoprotein hormones contain a common -glycoprotein subunit (GSU) that heterodimerizes with unique ß subunits ([1, 2] for reviews). In many teleost species, biochemical analysis [3–5] and molecular cloning studies [6–8] have demonstrated that there are two distinct GTHs: namely, GTH-I, which is homologous to FSH; and GTH-II, which is homologous to LH ([9] for a review). In salmonids, plasma levels of GTH-I are elevated during vitellogenesis and spermatogenesis; they decline during final maturation and ovulation in females and during spermiation in males. GTH-II levels are low during the early stages of the reproductive cycle; they increase during final maturation and ovulation in females and during spermiation in males [10]. Both GTHs are equipotent in stimulating estradiol-17ß (E2) production, but GTH-II is more potent than GTH-I in stimulating maturation-inducing steroid, DHP (17, 20ß-dihydroxy-pregnen-3-one [11]). These findings tend to suggest that, in salmonids, GTH-I plays a significant role in vitellogenesis and spermatogenesis, whereas GTH-II is important for final maturation and ovulation or spermiation; however, very little is known at present about the involvement of GTHs, especially GTH-I, in reproduction of fish species other than salmonids [12, 13].
Red seabream, Pagrus major (order: Perciformes), provides an interesting and unique model to study the physiological roles of GTHs in fish reproduction. In contrast to salmonids, red seabream have an asynchronous-type ovary and spawn almost every day during the spawning season. Previous reports from our laboratory have shown that, in vitro, the biological activity of GTH-I is much lower than that of GTH-II in production of E2 by vitellogenic follicles of red seabream [14], although GTH-I has a similar potency to that of GTH-II in stimulating in vitro 11-ketotestosterone (11-KT) production from testis slices of mature male fish [15]. In addition, more recent studies have demonstrated that GTH-II, not GTH-I, induces final maturation of oocytes of red seabream. These in vitro data raise the possibility that there may be functional differences between perciform and salmonid GTHs in regulation of reproduction, especially with the biological activity of GTH-I, and that sexual dimorphism may exist in the pattern of synthesis of GTHs between male and female red seabream.
In this study, to elucidate the roles of GTH-I and GTH-II in the reproduction of both male and female red seabream, the expression profile of GTH subunit genes was studied during sexual maturation using cDNA probes generated through a reverse transcription-polymerase chain reaction (RT-PCR) based cloning technique using degenerate oligonucleotide primers corresponding to the partial amino acid sequences of GTH subunits. We provide evidence showing a pattern of GTH subunit gene expression that is different from that of salmonids, and also a sexually dimorphic pattern of gene expression of Iß during sexual maturation in male and female red seabream.
MATERIALS AND METHODS
Animals
Red seabream, reared under natural conditions in net pens in Gokasho Bay, Nansei, Mie, Japan, were collected in February, March, April, and June 1997, times that corresponded to maturing (February–March), spawning (April–May), and regressed (June–July) phases. Fish were deeply anesthetized with 2-phenoxyehanol (Nacalai Tesque, Kyoto, Japan) before being killed by decapitation. The pituitary glands were removed and immediately frozen in liquid nitrogen and then stored individually at -80°C until extraction of mRNA. The body weight and length of each fish were recorded before dissection. The gonads were excised and weighed for calculation of the gonadosomatic index (GSI; [gonad weight/body weight] x 100%), then a small piece of the gonads was fixed in Bouins fixative for histological identification of maturation stage.
Amino Acid Sequences Analysis
Red seabream GTH-I and GTH-II were isolated from pituitary glands as previously described [5]. Separation of GSU and ß subunits was performed by reverse-phase (rp) HPLC [16]. Partial amino acid sequences of GTH subunits were determined by the method of Ito et al. [17]. Each of the purified subunits was treated with neuraminidase to remove sialic acid. Subsequently, the enzymatically modified subunits were reduced and carboxymethylated (RCM). Each RCM subunit was digested separately with several endopeptidases as follows: each RCM subunit (0.5 mg) was digested with lysyl endopeptidase (LE) at an enzyme-to-substrate ratio (E/S) of 1/60 (wt/wt) in 400 µl of 0.1 M ammonium bicarbonate buffer (pH 8.0) at 37°C for 4 h, with Staphylococcus aureus V8 protease (SP) at an E/S of 1/60 (wt/wt) in 200 µl of 0.05 M ammonium bicarbonate buffer (pH 8.0) at 37°C for 18 h, and with Pseudomonas fragi mutant metalloprotease (MP) at an E/S of 1/100 (wt/wt) in 300 µl of 0.05 M ammonium bicarbonate buffer (pH 8.0) at 37°C for 12 h. Fractionation of all fragments obtained from each RCM subunit was performed by rp HPLC on a TSK gel (Tosho, Tokyo, Japan) OSD-120T column (4.6 x 250 mm) with a linear gradient of isopropanol in 0.1% trifluoroacetic acid. The primary structures of peptides were determined by automated Edman degradation using an Applied Biosystem Model 471A protein sequencer with a Model 120A on-line phenylthiohydantoin analyzer. The sequence data were compared with known protein sequences in the EMBL and SwissProt protein database by the FASTA search program [18].
Oligonucleotides
Oligonucleotides (Custom Oligo Service, Tsukuba, Japan) used as PCR primers are listed here and shown in Figure 1. The degenerate PCR primers for GTH subunit cDNAs were designed from reverse translation of a partial amino acid sequence identified in this study. Primers for 18S rRNA were based on the nucleotide sequences of the other vertebrates [19].
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Primer 1. Degenerate primer (DP) for GSU: 5'-TAY CAR TGY ATG GGN TGY TG-3' (IUB group codes were used: R=A+G, S=C+G, Y=C+T, W=A+T, D=A+G +T, H=A+C+T, N=A+C+G+T).
Primer 2. DP for GSU: 5'-CAR TGR CAR TCN GTR TGR TT-3'.
Primer 3. GSU gene specific primer (-GSP) for 5'-RACE: 5'-TGG TTT CTC ACC CTT ATG CC-3'.
Primer 4. -GSP for 5'-RACE: 5'-TCA TAG CTG TGC TTT GCG AC-3'.
Primer 5. -GSP for 3'-RACE: 5'-TTC TCC AGA GCG TAC CCA AC-3'.
Primer 6. -GSP for full length: 5'-GTG AAT GAA GAG GGT ACT TTC TC-3'.
Primer 7. -GSP for full length: 5'-CAA TCT ACA GTT AAT CCT ATA CAT CTA TC-3'.
Primer 8. DP for Iß: 5'-ATG CCN GTN GAR WSN TGY GG-3'.
Primer 9. DP for Iß: 5'-GCN CAN GGR TAN GTD ATN GC-3'.
Primer 10. Iß gene specific primer (Iß-GSP) for 5'-RACE: 5'-TTC ACC TCA TAG GAC CAG TCC-3'.
Primer 11. Iß-GSP for 5'-RACE: 5'-GTC CTT TGT TCA GCC CAG TC 3-'.
Primer 12. Iß-GSP for 3'-RACE: 5'-AGC AAC GAG TTC ATC CAC AC-3'.
Primer 13. Iß-GSP for full length: 5'-GCT GGT ACA GAT GTT CAG AGA G-3'.
Primer 14. Iß-GSP for full length: 5'-TAA GTA CCA AGT TAC CAA AGA TG-3'.
Primer 15. DP for IIß: 5'-CAR YTN ATH AAY CAR ACN GT-3'.
Primer 16. DP for IIß: 5'-CRT TCA TRC ARA ART TNG G-3'.
Primer 17. IIß gene specific primer (IIß-GSP) for 5'-RACE: 5'-CTC AAA GGT GCA GTC AGA CG-3'.
Primer 18. IIß-GSP for 5'-RACE: 5'-ATG CTG ATA AAC GTA CCG GG-3'.
Primer 19. IIß-GSP for 3'-RACE: 5'-TGT CTC TTG AGA AGG AGG GC-3'.
Primer 20. IIß-GSP for full length: 5'-CAC ACT GCC TGC AGA TAA CAG-3'.
Primer 21. IIß-GSP for full length: 5'-TAC AAC AAT TGA CGC ACA GTT TC-3'.
Primer 22. Adapter primer: 5'-TGG AAG AAT TCG CGG CCG CAG-3'.
Primer 23. DP for 18S rRNA: 5'-AGA TAA CYT CGR GCC GAT CG-3'.
Primer 24. DP for 18S rRNA: 5'-CCA TCC AAT CGG TAG TAG CG-3'.
mRNA Isolation and Cloning of the Red Seabream GTH Subunit Genes
The pituitary gland was collected from a maturing female red seabream and total RNA was extracted from the gland (40 mg) according to the manufacturer's instructions (Pharmacia). Poly(A)+ RNA was isolated using a Pharmacia mRNA extraction kit.
Complemetary DNA was synthesized from 100 ng poly(A)+ RNA with NotI-d(T)18 bifunctional primer using the first-strand cDNA synthesis kit (Pharmacia Biotech, Uppsala, Sweden). An aliquot of the first-strand reaction was amplified with degenerate primers 1 and 2 for GSU, 8 and 9 for Iß, and 15 and 16 for IIß, respectively. PCR was performed in 50 µl final volume containing 5 µl of 10x reaction buffer, 2 mM MgCl2, 200 µM dNTP, 2 µM of each primer, and 2.5 U Taq DNA polymerase (Takara Biomedicals, Tokyo, Japan). After an initial 1.5-min denaturing step at 94°C, 30 cycles of amplification were performed using a cycle profile of 94°C for 30 sec, 50°C for 1 min, and 72°C for 1.5 min. After the last cycle, elongation was extended to 10 min at 72°C. PCR products were T-A cloned into pBluescript II KS- (Stratagene, La Jolla, CA) and sequenced.
5'-RACE was performed using a 5'-RACE kit (Gibco/BRL, Grand Island, NY) according to the manufacturer's instructions. Briefly, 100 ng of red seabream pituitary mRNA was reverse-transcribed using 20 units of Superscript II with each specific primer (GSU: primer 3, Iß: primer 10, IIß: primer 17). The cDNA was C-tailed and then PCR-amplified using a second specific primer for each (GSU: primer 4, Iß: primer 11, IIß: primer 18) and 5'-RACE abridged anchor primer. The PCR-amplified DNA fragments were fractionated in a 4% agarose gel, subcloned into pBluescript II KS- (Stratagene), and sequenced as just described.
For 3'-RACE, 100 ng of the pituitary poly(A)+ RNA was reverse-transcribed using 50 pmol of NotI-d(T)18 bifunctional primer by 200 units of MLVE (Pharmacia), followed by PCR between the primers (primers 5 and 22 for GSU, primers 12 and 22 for Iß, and primers 19 and 22 for IIß). PCR was initiated at 94°C for 30 sec, followed by 30 cycles of amplification under conditions of 94°C for 30 sec, 50°C for 1 min, and 72°C for 1.5 min. After the last cycle, elongation was extended to 10 min at 72°C. The resultant DNA fragment was ligated and subcloned using pBluescript II KS- (Stratagene), and sequenced. The full-length cDNA was generated by PCR using ExTaq polymerase (Takara) with the same first-strand cDNA and primers 6 and 7 for GSU, primers 13 and 14 for Iß, and primers 20 and 21 for IIß.
Cloning of Red Seabream 18S rRNA
Genomic DNA from red seabream was isolated by homogenizing the liver of a female red seabream and subsequently treating it with proteinase [20]. Using genomic DNA (100 ng) as a template, PCR was performed with degenerate primers 23 and 24. PCR conditions and subcloning into the plasmid vector pBluescript II KS- were as described earlier.
Nucleotide Sequencing Analysis
DNA was sequenced in both strands by the dideoxynucleotide chain-termination method [21] using a dRhodamine terminator cycle sequencing kit (Perkin-Elmer/Applied Biosystems, Chiba, Japan) and an Applied Biosystems Model 377 DNA sequencer. At least four independent clones were sequenced by the dideoxy chain-termination method for each GTH subunit to eliminate misincorporation of nucleotides with Taq DNA polymerase. The nucleotide and amino acid sequences were analyzed using Geneworks software (release 2.45; IntelliGenetics, Inc., Mountain View, CA) and the BLAST network service of the National Center for Biotechnology Information [22]. The sequences discussed in this paper have been submitted to DDBJ/EMBL/GenBank (accession numbers AB028211, AB028212, AB028213, and AB028214).
Northern Blot Analysis
Three pituitary glands were isolated from fish at different maturational stages and pooled. Three replicate samples were made for each stage (i.e., 3 samples of 3 pooled pituitary glands). Total RNA from pooled sample was isolated by the guanidium isothiocyanate method [23]. Fifteen micrograms of total RNA per sample were denatured at 65°C for 15 min in 50% formaldehyde and subjected to electrophoresis on a 1% agarose gel in 0.2 M MOPS, pH 7.0, containing 2.2 M formamide, 50% formaldehyde, and then transferred onto a Hybond N+ nylon membrane (Amersham Pharmacia Biotech). The membrane was air-dried and baked at 80°C for 2 h before hybridization with radiolabeled full-length red seabream GTH subunit cDNA probes. The membrane filter was washed at 65°C with several buffer changes of decreasing SSC concentrations from 2x to 0.1x (1x SCC = 0.1 M NaCl and 0.015 M sodium citrate). The radioactivity of each hybridization signals was counted using a BetaScope Model 603 Blot Analyzer (Aloka, Tokyo, Japan). Then, under the same conditions, the blot was rehybridized with 18S rRNA to control for loading variations. All values for mRNA levels were arbitrary radioactive units after standardizing with the levels of 18S rRNA. To check the specificity of hybridization signals, 0.1–100 pg of unlabeled plasmid DNA encoding red seabream GTH subunits were spotted on a nylon membrane and allowed to react with the same specific probes that were used for Northern blot analysis. The results indicated negligible cross-hybridization among the probes.
Statistical Analysis
All data were expressed as the mean ± SEM. Seasonal variation in the levels of the GTH subunits mRNAs in the pituitary of red seabream were examined by one-way ANOVA followed by Duncan's multiple-range test. GSI data were analyzed by the Kruskal-Wallis test.
RESULTS
Primary Structure of GTH Subunits
The amino acid sequence of GSU is summarized in Figure 2. The solid line indicates the residues determined by the automated Edman degradation. The GSUs of GTH-I and GTH-II were digested separately by LE. Sequence analysis of the LE-digested RCM-GSU of GTH-I resulted in determination of 76 residues, except for the 40th to 48th, 55th, and 87th to 94th. The amino acid sequence of GTH-II GSU was determined by the same procedure that was used for GTH-I GSU. Comparison of peptide maps of GTH-II GSU with those of GTH-I GSU indicated that GTH-II GSU was identical to GTH-I GSU in amino acid sequence (data not shown). The entire RCM-Iß molecule was first evaluated to clarify the sequence from the N-terminal to the 42nd residue. Following sequence analysis of one LE, four SP, and six MP peptides, three residues (52nd, 53rd, and 102nd) remained undetermined (Fig. 3.). The N-terminal amino acid sequences of IIß was determined up to the 40th residue. With amino acid sequences of LE, SP, and MP fragments, the complete amino acid sequence of IIß can be proposed as summarized in Figure 4.
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Isolation and Characterization of cDNA Encoding Red Seabream GSU
A 629-base pair (bp) cDNA encoding GSU was isolated by RT-PCR from a red seabream pituitary gland. During the first experiment, a fragment of ~200 bp amplified with a degenerate pair of primers 1 and 2 (see Materials and Methods) designed from the reverse translation of a partial peptide sequence. The deduced amino acid sequence of this cDNA fragment agreed with the partial amino acid sequence of GSU. In the second step of cloning, gene-specific primers of identified sequences were used for 5'- and 3'-RACE, and the full cDNA sequence was determined. Subsequently, the full-length cDNA was amplified with a pair of primers targeted to the 5' and 3' ends, cloned, and sequenced.
The nucleotide sequence of GSU was derived from an open reading frame (ORF) of 351 bp, which encoded a protein of a calculated molecular weight of 13.1 kDa (Fig. 2.). The coding region was followed by a 3'-untranslated region (3'-UTR) of 229 bp that contained a polyadenylation signal, ATTAAA, 8 bp upstream from the poly(A)+ tail. Northern blot analysis shows that one transcript of approximately 720 bp is present in red seabream pituitary (Fig. 5). Figure 6 shows the deduced amino acid sequence of the red seabream GSU cDNA aligned with counterparts from other species. The red seabream GSU shares 94% and 95% overall sequence identity with bonito and tuna, respectively; species that belong to the same order (Perciformes); 63%–73% GSUs of other fishes; and 63% and 56% with those of rat and human, respectively. The positions of ten cysteines were conserved. In addition, two consensus N-linked glycosylation sites (N-X-S/T) were identified at Asn55 and Asn80. On the basis of a comparison with N-terminal amino acid purified GSU of red seabream and from studies by von Heijne [24, 25], the cleavage site of the signal peptide was determined to be between Ser (a.a.-1) and Tyr (a.a.+1).
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Isolation and Characterization of cDNAs Encoding Red Seabream ß Subunits
The cDNAs encoding mature ß subunit peptides were amplified from the red seabream pituitary mRNA using RT-PCR. During the first experiment, partial ß subunit cDNAs (Iß: ~200 bp; IIß: ~300 bp) were amplified with each degenerated pair of primers designed according to the partial amino acid sequences of Iß and IIß subunits (see Materials and Methods). The sequences deduced from isolated cDNA fragments agreed with the partial amino acid sequences of ß subunits. In the second step, by means of 5'- and 3'-RACE of newly identified sequences, the remaining sequence information was determined. Finally, the full-length cDNAs were amplified with a pair of primers targeted to the 5'- and 3'-ends, cloned, and sequenced. The nucleotide and deduced amino acid sequences of Iß and IIß subunits are shown in Figures 3 and 4, respectively. The Iß cDNA consisted of 531 bp and included an ORF encoding a protein of 120 amino acids in length with a calculated molecular weight of 13.0 kDa. The 3'-UTR was 76 bp in length and contained two polyadenylation signals; 3 bp and 10 bp upstream from the poly(A)+ tail. The 557-bp IIß cDNA comprised an initiation codon (ATG: 28 bp), an ORF (465 bp), a stop codon (TAA), and the 3'-UTR, including a polyadenylation signal 12 bp upstream of the poly(A)+ tail. The molecular weight of the protein encoded by this gene was 16.3 kDa. Northern blot analysis (Fig. 5) shows the mRNA transcripts of Iß and IIß subunits were approximately 700 bp and 720 bp, respectively. The alignment of the deduced amino acid sequence of the red seabream ß subunits with other forms of ß subunits is shown in Figure 6. The red seabream Iß shared a high homology with bonito (68%) and tuna (67%), 37%–45% with other teleosts and 29% and 30% with rat and human, respectively. The positions of twelve cysteines and one N-linked glycosylation site (Asn9) were completely conserved. For the IIß sequence, the highest homology was shared with bonito (80%) and tuna (80%); 56%–67% with other teleosts; and 36% and 37% with rat and human, respectively. The positions of twelve cysteines and one N-linked glycosylation site (Asn20) were completely conserved. Based on the N-terminal amino acid sequences and from studies by von Heijne [24, 25], cleavage sites appear to be between Ser (a.a.-1) and Typ (a.a.+1) for Iß, and between Try (a.a.-1) and Cys (a.a.+1) for IIß.
Seasonal Changes in mRNA Levels of GTH Subunits in the Red Seabream at Various Stages of Sexual Maturation
Changes in GSI of male and female red seabream sampled from February to June 1997 are shown in Figure 7. GSIs of both males and females gradually increased from February and reached a peak during the spawning phase, in April. Histological examination revealed that previtellogenic oocytes (oil droplet stage) were present in the ovaries of female red seabream collected in February, when GSI was 1.51 ± 0.31% (Fig. 8A). Vitellogenic oocytes were first observed in the ovaries of fish collected in March with a GSI of 2.88 ± 0.48% (Fig. 8B). In April, oocytes were observed at various developmental stages from perinucleolus to mature stage (Fig. 8C). At this time, the GSI was 7.08 ± 1.31%. In June, when GSI was 0.49 ± 0.06%, only perinucleolus-stage oocytes were found in the ovary (Fig. 8D). In male fish, the presence of all stages of germ cells in the testes collected from February (GSI: 1.39 ± 0.27%) to April (GSI: 7.24 ± 0.81%) (Fig. 8, E–G). In February and March with a GSI of 2.88 ± 0.48%, spermatocytes and spermatids predominantly occupied the testes of the fish (Fig. 8, E and F). As shown in Figure 8G, spermatozoa were predominantly occupied in April; however, spermatogonia were the only germ cells present in the testes in June with a GSI of 0.28 ± 0.03% (Fig. 8H).
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Expression of GTH subunit genes during sexual maturation was assessed by Northern blot analysis and levels of 18S rRNA were used for internal standardization. There were no significant changes observed in 18S rRNA levels during sexual maturation, indicating that 18S rRNA is an appropriate internal standard in these experiments. Figure 9A shows the changes in mRNA levels of GSU in males and females. In males, the levels of GSU mRNA gradually increased from February and reached a maximum in April. Similarly, the levels of GSU mRNA of females were high from February to April. Subsequently, GSU mRNA levels in both males and females significantly decreased in June. A one-way ANOVA followed by a Duncan's multiple range test revealed that significant differences (P < 0.01) between sexes in February, April, and June. Iß mRNA levels in males gradually increased from February to March, reached a peak in April, and then declined in June (Fig. 9B). In contrast, Iß mRNA levels in females were found to be considerably lower than those of males over the entire sampling period. Statistical analysis revealed that mRNA levels of Iß in both sexes in April (the spawning phase) were significantly different (P < 0.05) from those in any other months studied (Fig. 9B). Conversely, mRNA levels of IIß in both males and females were high from February to April, decreased in June, and no significant differences were observed between levels in males and females during the experiment period (Fig. 9C).
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DISCUSSION
In this study, the cDNAs encoding red seabream GSU and GTH ß subunits were cloned and their nucleotide sequences determined. We were then able to describe the expression of GTH subunit genes in males and females during sexual maturation.
The deduced amino acid sequence of mature GSU shares a high homology with those of other teleosts. The positions of all ten cysteines and two putative N-glycosylation sites are well conserved. From a comparison with amino acid sequences of other vertebrates, it appears that one region is highly conserved; this region is located between amino acids 33 and 66, and consists of two paired adjacent cysteines and the first putative N-linked glycosylation site. This region of GSU is believed to be partially involved, totally involved, or both in subunit assembly, receptor binding, or both as suggested in human GSU [26]. In this study, only one type of cDNA for GSU was cloned from red seabream pituitary. There is only one type of GSU in pituitaries of higher vertebrates [27], whereas two types of GSU exist in several tetraploid teleosts [7, 28]. Southern blot analysis showed that the red seabream genome contained only one copy of the GSU gene (data not shown); thus, red seabream has only one type of GSU in the pituitary. This is further supported by the peptide map showing that GTH-I GSU and GTH-II GSU are identical.
High sequence homology was observed between the deduced amino acid sequence of mature Iß and IIß subunits of red seabream and their counterparts from other teleosts, especially perciform species. Furthermore, the position of all cysteines, which are related to the folding tendency of the protein and the rigid conformation of the GTH family, are well conserved. Putative N-linked glycosylation sites are also conserved among teleosts. (Fig. 6). Because the glycosylation of human FSH is important for the coupling of the receptor to adenylate cyclase [29], the sugar chain in teleost Iß may also be involved in receptor binding or signal transduction mechanisms. Multiple protein sequence alignments of LHß/IIß also showed that red seabream IIß had an extended N-terminus (Fig. 6). Although deduced amino acid sequences have been described for many teleosts, to our knowledge, there is little information about amino acid sequences of IIß obtained by biochemical analyses [13, 14, 30]. Efforts to analyze the N-terminus of IIß in other teleosts will help to resolve knowledge of the molecular evolution of red seabream IIß.
Northern blot analysis of red seabream pituitary total RNA revealed a single mRNA transcript of about 720 bp for GSU, 700 bp for Iß, and 720 bp for IIß (Fig. 5). The size of each mRNA transcript of GTH subunits was longer than the respective cloned cDNA sequences. This discrepancy can be attributed to the length of the poly(A)+ tail. Alternatively, it is possible that the 5'-UTRs of the subunits were are not completely cloned. Further work is required to confirm the 5'-UTR results using a different approach, such as a primer extension, S1 nuclease protection analysis, or both.
According to Northern blot analysis, Iß mRNA levels of males increase in association with an increase in GSI levels and gonadal development during sexual maturation. This is consistent with findings reported for rainbow trout, in which Iß mRNA levels predominate in early stages of gonadal development [31]. Furthermore, GTH-I stimulates in vitro production of 11-KT, a main androgen in teleosts [32] by testes of male red seabream [15]. Taken together, these results lend support to the hypothesis that in male red seabream, GTH-I has an important role in spermatogenesis, similar to that of salmonids. In contrast to salmonids, the Iß mRNA levels of red seabream were still high in the spawning season. Because spermatogenesis occurs in the spawning season simultaneously with spermiation, high levels of Iß in this season are not unusual. IIß mRNA levels are high from early spermatogenesis to the spawning season in red seabream. This is completely different from the data obtained in salmonids, in which IIß mRNA levels are very low during early gametogenesis and increase rapidly during late gametogenesis [31]. Similar to GTH-I, in vitro, GTH-II stimulated 11-KT production in testis slices of male red seabream [15]. These data, together with the present data, suggest that GTH-II is also involved in spermatogenesis in this fish. Unlike salmonids, in this study, all developmental stages of gametes in red seabream (from spermatogonia to spermatozoa) were present in the testes used; thus, we could not conclude that there are differences between two GTHs in their physiological roles in spermatogenesis and spermiation in male red seabream.
In contrast to males, the Iß mRNA levels of females were maintained at low levels during sexual maturation, although levels increased slightly in the spawning season. The expression pattern of Iß mRNA in female red seabream is different from that reported in salmonids [31], in which Iß mRNA levels and the plasma levels of GTH-I predominate during the initial phase of vitellogenesis [31, 33]. Moreover, the biological activity of GTH-I is much lower than that of GTH-II in in vitro E2 production of vitellogenic follicles [14] and GTH-I is ineffective in the induction of germinal vesicle break down (GVBD; [15]); therefore, unlike salmonids, GTH-I may not have any significant roles in steroidogenesis and gonadal development of female red seabream. GTH-I has been shown to increase the rate of vitellogenin uptake by the oocyte of rainbow trout in vitro and in vivo [34]; hence, it may be interesting to examine whether GTH-I has additional roles, such as uptake of vitellogenin into oocytes of female red seabream.
Currently, it remains unclear whether the pattern of Iß mRNA levels is correlated with that of plasma GTH-I levels, because there is no specific radioimmunoassay available to measure plasma GTH-I levels in red seabream. Immunocytochemical analysis, however, shows that the number of GTH-I immunoreactive cells increases during sexual maturation in male red seabream [16], suggesting that Iß mRNA levels in the pituitary may correlate with those of the protein. This is consistent with observations that Iß mRNA levels and plasma GTH-I levels rise in parallel during gametogenesis in salmonids [35]; therefore, plasma GTH-I levels may positively correlate with the expression of Iß subunit genes in the pituitary of red seabream during sexual maturation.
This study also demonstrates that IIß mRNA levels are high from the beginning of early vitellogenesis to the spawning season in female red seabream. A previous study showed that plasma levels of GTH-II increased with gonadal development and reached a peak during spawning season in female red seabream [16]. These results are different from the pattern of changes observed in plasma levels of GTH-II in the rainbow trout [31] and striped bass [36]. In these species, IIß levels are low or nondetectable during vitellogenesis and increase at final oocyte maturation. In female red seabream, GTH-II is more potent than GTH-I in stimulating E2 production by vitellogenic oocytes [14]. Moreover, GTH-II, but not GTH-I, induced final maturation of red seabream oocytes [15]. Plasma GTH-II increased in association with the final maturation and ovulation induced by injection of a gonadotropin-releasing hormone analogue [5] and a daily rhythm of increased GTH-II levels linked to oocyte maturation was observed in females that spawned naturally during the spawning season [16]. Thus, these data suggest that GTH-II is also involved in final oocyte maturation in red seabream. In female red seabream, GTH-II may have important roles both in final maturation of oocytes and, unlike salmonids, in vitellogenesis. African catfish (Clarias gariepinus) GTH-I-like hormone could not be isolated and only one type of gonadotroph is present in the pituitary [37, 38]; thus, it is assumed that GTH-II performs a major role in female reproduction [39] similar to that proposed for red seabream.
In both sexes, the GSU mRNA levels were high during vitellogenesis and spermatogenesis and at the spawning phase, then sharply declined in the regressed phase, although, overall, mRNA levels in males were higher than those in females. In situ hybridization shows that an increase in mRNA levels of GSU is correlated with active transcription of the GSU gene in the gonadotrophs of the rainbow trout [35]. Although it has not yet been analyzed, it is likely that the increase in GSU mRNA levels detected in our study is the result of enhanced gene transcription in gonadotrophs. However, because GSU mRNA is potentially expressed in both gonadotrophs and thyrotrophs, we can not rule out the possibility that thyrotrophs contribute to the observed increases in GSU mRNA levels.
This study clearly shows sexual dimorphism in Iß gene expression during sexual maturation in the red seabream. These data are in agreement with previous data showing that, in gilthead seabream, Iß mRNA levels were 2.5-fold higher in males compared with females during the spawning phase [40]. The mechanisms underlying gender-specific patterns of Iß gene expression in the pituitary of male and female red seabream are currently unclear. The expression of GTH subunit genes are regulated by various endocrine factors, including hypothalamic releasing hormones [36, 41] and gonadal steroids [42–44]. In the male rat, androgens positively regulate higher steady-state levels of FSHß expression, but not GSU or LHß, than in females [45, 46]. More recently, Borg et al. [47] showed that 11-ketoandrostenedione increases the pituitary and plasma GTH-I levels in male Atlantic salmon; thus, it is possible that sex steroids may be involved in sexual dimorphism in gene expression of Iß in red seabream. Therefore, additional studies, including castration and steroid hormone replacement, and primary pituitary cultures obtained from male and female red seabream, would be helpful to draw more definite conclusions.
ACKNOWLEDGMENTS
The authors thank Mr. T. Okura for technical assistance and Drs. G. Young (University of Otago) and M.S. Alam for their critical reading of the manuscript.
FOOTNOTES
First decision: 20 December 1999.
1 Financially supported in part by a grant-in-aid from the Ministry of Agriculture, Forestry and Fisheries (ED-00-11-3-1). 
2 Correspondence. FAX: 81 596 58 6413; kgen{at}ss.nria-tmk.affrc.go.jp
Accepted: February 3, 2000.
Received: November 29, 1999.
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